Inhibition of ligand-mediated HER2 activation in androgen-independent prostate cancer

Hormone-independent tumor growth and metastasis are associated with increased mortality in human prostate cancer. In this study, we evaluate a potential role for ligand-mediated activation of HER2 receptor tyrosine kinase in androgen-independent prostate cancers. HER2, HER3, and epidermal growth factor receptor were detected in the androgen-independent cell line 22Rv1. Heregulin stimulation results in receptor phosphorylation and cell proliferation that is inhibited by increasing concentrations of anti-HER2 recombinant humanized monoclonal antibody (rhuMAb) 2C4. Furthermore, inhibition of tumor growth was observed in xenografts derived from 22Rv1 cells when treated with rhuMAb 2C4 in a dose-dependent manner. These studies provide a framework, both in vitro and in vivo, to examine the molecular mechanisms of ligand-driven HER2 activation in androgen-independent tumorigenesis.     

Tumor retention of 186Re-MAG3, 111In-DTPA and 125I labeled monoclonal antibody G250 in nude mice with renal cell carcinoma xenografts

In radioimmunotherapy of solid tumors substantial gain might be achieved by carefully selecting the radionuclide and the linker connecting it to the antibody. In contrast to 131I, radiometals such as 90Y and 111In may be retained in the tumor cell after internalization of the antibody, thereby enhancing the radiation dose to the tumor. The physical properties of 186Re with 1.08 MeV beta-emission (71%) and 137 keV gamma-emission (10%) seem ideal for radioimmunotherapy. In this study we investigated in nude mice with s.c. renal cell carcinoma xenografts the biodistribution and the retention in the tumor of 186Re-MAG3 labeled monoclonal antibody (mAb) G250 as compared to 111In-DTPA-G250, to 125I-G250 and to 131I-MN14 (non-specific control mAb). Radiolabeled antibody preparations were i.v. injected. Seventy two hours p.i. the biodistribution of the radiolabel was determined. Blood levels of all mAb G250 preparations were remarkably low (mean: 2.38, 1.40 and 1.43% ID/g for 125I, 111In and 186Re respectively) whereas blood levels of mAb MN14 were significantly higher (mean: 12.3% ID/g), indicating tumor processing of mAb G250. Retention of 111In in the tumor was significantly higher than of 186Re and 125I whereas retention in the tumor of 186Re and 125I did not differ significantly. CONCLUSION: In contrast to other radiometals such as 111In and 90Y, 186Re is not retained in the tumor cell. Therefore 186Re has no additional advantage for radioimmunotherapy with respect to retention in the tumor.     

Circulating HER2 extracellular domain and resistance to chemotherapy in advanced breast cancer.

To test the hypothesis of an association between HER2 and chemotherapy resistance, we performed a prospective assessment of the predictive value of the circulating HER2 extracellular domain (ECD) in patients with advanced breast carcinoma in the setting of a multicenter Phase II trial using paclitaxel and doxorubicin. Serum samples were collected from 58 patients with metastatic breast carcinoma before first-line chemotherapy for advanced disease, and the levels of circulating HER2 ECD were measured using an enzyme immunoassay. Immunohistochemistry with anti-HER2 monoclonal antibody CB11 was used to assess the overexpression of HER2 in the primary tumors. When 450 fmol/ml was used as a cutoff, 24 cases (41%) had elevated HER2 ECD levels. Elevated levels of circulating HER2 ECD were associated with the expression of HER2 in the primary tumor tissue and with the metastatic tumor burden (evaluated with the marker CA 15-3; P = 0.032 and P = 0.002, respectively) but not with variables such as menopausal status, stage at diagnosis, previous adjuvant therapy, or the number of metastatic sites. The levels of circulating HER2 ECD correlated inversely with the response to treatment. The probability of obtaining a complete response to chemotherapy was significantly lower (P = 0.021) in patients with elevated HER2 ECD levels (0%; 95% confidence interval, 0-13%) compared with patients with nonelevated HER2 (26%; 95% confidence interval, 12-45%). In addition, the duration of clinical response was significantly shorter in patients with elevated HER2 ECD, compared with the cases with nonelevated HER2 (7.5 versus 11 months; P = 0.035). In conclusion, elevated levels of circulating HER2 ECD in patients with metastatic breast cancer correlate with reduced efficacy of a paclitaxel-doxorubicin chemotherapy combination. We suggest that the poor response rate associated with HER2 expression in advanced breast cancer may not be reversed by aggressive chemotherapy alone.     

Biweekly gemcitabine plus vinorelbine in first-line metastatic breast cancer: efficacy and correlation with HER2 extracellular domain

Purpose To assess the toxicity and efficacy of biweekly gemcitabine plus vinorelbine in first-line advanced breast cancer, and to establish whether circulating HER2 ECD levels correlate with the efficacy of the combination. Patients and methods 52 patients were treated with gemcitabine 2500 mg/m² plus vinorelbine 30 mg/m², both on day 1 of 14-day cycles, for a maximum of 10 cycles. Baseline serum levels of HER2 ECD were assessed with an ELISA. Results All patients were evaluable for toxicity, and 50 for efficacy. Overall toxicity was moderate. Grade 3 neutropenia occurred in 35% of patients and grade 4 in 19%. Other grade 3 toxicities were observed in less than 6%. There was one episode of febrile neutropenia, and one death after cycle three. Overall response rate was 52% (95% CI: 38% to 66%), with 2 patients achieving a CR (4%). Response rate did not correlate with HER2 ECD, with 50% of HER2 ECD positive patients responding, vs 48.5% of the HER2 ECD negative. Median overall survival was 24.6 months. Conclusion Gemcitabine plus vinorelbine, given as an every-two-week schedule, is an active regimen in advanced breast carcinoma. This combination can be an option when anthracyclines and taxanes are not preferred. HER2 ECD has no predictive value in this non-taxane combination.     

Herceptin and its therapeutic strategy in patients with breast cancer overexpressing HER2

Herceptin (trastuzumab) is a recombinant humanized murine monoclonal antibody that recognizes HER2/neu cell-surface receptors and has been shown to be active both in combination with adriamycin (epirubicin)/cyclophosphamide or taxanes and as a single agent, either in the 1st or 2nd/3rd line treatment of women with metastatic breast cancer with HER2 overexpression by IHC or gene amplification by FISH. Preliminary results of Herceptin with other agents such as vinorelbine, cisplatin and various hormonal agents are also promising, and these combinations warrant further clinical exploration. Large-scale multicenter trials including a European and an international study in adjuvant setting have started for high-risk women with HER2 overexpressing breast cancer, with total planned recruitment of nearly 10,000 women.     

Targeting c-erbB2 and other receptors of the c-erbB family: rationale and clinical applications

The c-erbB family of receptors includes four distinct receptors, namely c-erb B1, 2, 3 and 4 (HER1, 2, 3 and 4, respectively). Trastuzumab (T) is a recombinant humanized anti-HER2 monoclonal antibody that binds the extracellular domain of the receptor and blocks intracellular signalling. In clinical studies of T, either alone or in combination with chemotherapy, in HER2 overexpressing metastatic breast cancer patients, a significant benefit was obtained--improved response rates and survival, when T was combined with chemotherapy. Several trials of adjuvant T, either singly or in combination with chemotherapy, are in progress in early breast cancer patients. Pertuzumab defines a new class of HER2 inhibitors, "dimerization inhibitors" that block both homo- and hetero-dimerization of HER2. In preclinical studies pertuzumab is inhibitory to breast, prostate and non small cell lung cancer cell lines, both over and non overexpressing HER2. In phase I clinical trials pertuzumab has shown activity in a number of human cancers. A phase II program is in progress.     

In vitro characterization of a bivalent anti-HER-2 affibody with potential for radionuclide-based diagnostics

The 185 kDa transmembrane glycoprotein human epidermal growth factor receptor 2 (HER-2) (p185/neu, c-ErbB-2) is overexpressed in breast and ovarian cancers. Overexpression in breast cancer correlates with poor patient prognosis, and visualization of HER-2 expression might provide valuable diagnostic information influencing patient management. We have previously described the generation of a new type of affinity ligand, a 58-amino-acid affibody (Z(HER2:4)) with specific binding to HER-2. In order to benefit from avidity effects, we have created a bivalent form of the affibody ligand, (Z(HER2:4))2. The monovalent and bivalent ligands were compared in various assays. The new bivalent affibody has a molecular weight of 15.6 kDa and an apparent affinity (K(D)) against HER-2 of 3 nM. After radioiodination, using the linker molecule N-succinimidyl p-(trimethylstannyl) benzoate (SPMB), in vitro binding assays showed specific binding to HER-2 overexpressing cells. Internalization of 125I was shown after delivery with both the monovalent and the bivalent affibody. The cellular retention of 125I was longer after delivery with the bivalent affibody when compared to delivery with the monovalent affibody. With approximately the same affinity as the monoclonal antibody trastuzumab (Herceptin) but only one tenth of the size, this new bivalent molecule is a promising candidate for radionuclide-based detection of HER-2 expression in tumors. 125I was used in this study as a surrogate marker for the diagnostically relevant radioisotopes 123I for single photon emission computed tomography (SPECT)/gamma-camera imaging and 124I for positron emission tomography (PET).     

Characterization of novel murine monoclonal antibodies directed against the extracellular domain of human HER2 tyrosine kinase receptor

HER2 proto-oncogene encodes a transmembrane receptor tyrosine kinase overexpressed in a variety of solid tumors. Several mouse monoclonal antibodies (MAbs) have been developed that recognize the extracellular part of HER2; of them two MAbs were humanized and employed for targeted immunotherapy. In this study we aimed to produce murine MAbs that specifically recognize the extracellular domain of human HER2. BALB/c mice were first primed with HER2-transfected NIH-3T3 cells and then boosted with recombinant extracellular part of HER2. Splenocytes from hyperimmunized mice were fused with myeloma cells and growing hybridomas were selected and screened for HER2 reactivity by an indirect ELISA. HER2-specific hybridomas were selected, cloned by limiting dilution assay, and further characterized by Western blotting and flow cytometry techniques. All clones showed positive reactivity to HER2 with binding affinity, ranging from 1.9×10(8) to 5×10(9), and stained HER2-transfected cells and malignant cells overexpressing HER2. None of the MAbs inhibited the binding of trastuzumab (Herceptin(?)) to HER2, indicating recognition of distinct epitopes by these MAbs. Based on these findings, our MAbs could be potentially used for selective targeting of HER2-expressing malignancies.     

Trastuzumab and lapatinib modulation of HER2 tyrosine/threonine phosphorylation and cell signaling

Cell surface transmembrane signaling receptors EGFR, HER3, and HER4 are activated by ligand-binding-mediated dimerization and phosphorylation. In contrast, HER2 amplification promotes signaling by increasing homo/heterodimerization and ligand binding. Trastuzumab or lapatinib therapy of HER2 amplicon-positive breast cancer cells induces growth inhibition and intracellular growth pathway signaling modulation. The mechanism(s) by which trastuzumab, an IgG1 humanized antibody, induces modification of cell signaling upon binding to an extracellular determinant on a ligand-less ??receptor?? membrane protein remains unexplained. Using immune detection methodology comprised of antibodies detecting three distinct domains of HER and five tyrosine/threonine phosphorylation sites, the effects of trastuzumab and lapatinib were defined during steady state growth inhibition. Here, we show that lapatinib markedly reduces HER2 tyrosine phosphorylation, while in contrast, no change in tyrosine phosphate levels is detected during trastuzumab-mediated cell growth inhibition. As trastuzumab treatment does not change either the steady state HER2 protein levels or HER2 mRNA, these findings argue against an antibody-dependent alteration in internalization kinetics. We further show a sequenced relationship between lapatinib-induced blockage of phosphorylation (6?C8?h) and induction of delayed cell death (5?C6?days), while trastuzumab-treated cells showed no evidence of cell death up to 9?days. Taken together, these results demonstrate that inhibition of HER2 phosphorylation by lapatinib is sufficient to induce apoptosis while trastuzumab binding to the extracellular HER2 domain may function by sterically modulating the detection of phosphate moieties by cytoplasmic signal transducers. This investigation also detected a 20?kD protein, which is down-regulated by lapatinib, further demonstrating the complexity of this signal transduction system.     

Estrogen-dependent,tamoxifen-resistant tumorigenic growth of MCF-7 cells transfected with HER2/neu

Summary Since the poor prognosis associated with HER2 amplified breast cancers might be explained by a mechanistic association between p185^HER2 overexpression and therapeutic resistance, we assessed the chemo-endocrine sensitivity of estrogen receptor (ER) containing MCF-7 breast cancer cells transfected with full-length HER2 cDNA. Of the 36 isolated MCF/HER2 subclones, 7 were found to overexpress p185^HER2 surface receptor at levels 3 to 45-fold greater than parental or control transfected cells (MCF/neo). The overexpressing transfectants possessed increased inositol-1,4,5-trisphosphate-3'-kinase activity comparable to enzyme activity in the endogenously HER2 amplified breast cancer cell lines SK-Br-3 and BT-474. The anti-p185^HER2 monoclonal antibody and receptor-specific partial agonist, muMAb4D5 (4D5), known to inhibit growth of SK-Br-3 and BT-474 cells, produced no significant growth inhibitory effect on any of the transfectants including the 45-fold overexpressing MCF/HER2–18 cells which were studied in greater detail. MCF/HER2–18 cells contained at least partially functioning exogenous receptor since 4D5 (3µg/ml) specifically stimulated phosphorylation of p185^HER2 and its co-precipitating ptyr56 substrate within 5 min, and this was followed at 1 h by a transient induction ofc-myc but notc-fos mRNA. ER content and thein vitro sensitivity of MCF/HER2–18 cells to 5-fluorouracil and adriamycin were identical to those of control transfectants and parental cells. However, these highly overexpressing transfectants had acquired low level (2 to 4-fold) resistance to cisplatin and were no longer sensitive to the antiestrogen tamoxifen (TAM). To compare the hormone-dependent tumorigenicity of the HER2 transfectants, MCF/HER2–18 and control cells (MCF, MCF/neo-3) were implanted into ovariectomized athymic nude mice. No tumors were produced in the absence of estradiol (E₂) administration. In E₂ supplemented mice, MCF/HER2–18 tumors grew most rapidly. When E₂ treatment was stopped and daily TAM injections were initiated, MCF-7 and MCF/neo-3 tumor growth ceased immediately, while MCF/HER2–18 tumors continued to show an accelerated growth rate lasting weeks. This pattern of hormone-dependent, TAM-resistant growth exhibited by the MCF/HER2–18 tumors in nude mice supports the possibility that p185^HER2 overexpression in human breast cancers may be linked to therapeutic resistance.     

Epitope mapping of human HER2 specific mouse monoclonal antibodies using recombinant extracellular subdomains of HER2

Background: Human epidermal growth factor receptor 2 (HER2) is overexpresed in several human malignancies. Numerous studies show that HER2 plays an important role in the development and maintenance of the malignant phenotype. Targetting of HER2 molecule by monoclonal antibodies (mAbs) is a promising therapeutic approach. Anti-HER2 mAbs affect cancer cells differently, targeting distinct epitopes of HER2. Methods: Reactivity of a panel of 8 mouse anti-HER2 mAbs was investigated by ELISA and Western blotting using different subdomains of the extracellular domain (ECD) of HER2. All subdomains of HER2, including I, II, III, IV, I+II, III+IV and full HER2-ECD were constructed and expressed in CHO cells. Cross-reactivity of the mAbs with other members of the human HER family and Cynomolgus HER2 was also studied by ELISA. The mAbs were also tested by immunohistochemistry (IHC) using HER2 positive breast cancer tissues. Results: Our results demonstrated that 3 out of 8 mAbs detected conformational epitopes (1T0, 2A8 and 1B5), while 5 mAbs identified linear epitopes (1F2, 1H9, 4C7, 1H6 and 2A9). Three of the mAbs recognized subdomain I, one mAb reacted with subdomain I+II, 2 mAbs recognized either subdomain III or IV and 2 mAbs recognized subdomain III+IV. However, none of our mAbs recognized subdomain II of HER2 alone. The mAbs displayed either inhibitory or stimulatory effect on HER2-overexpressing tumor cells and did not react with other members of the human HER family. The pattern of IHC results implies better reactivity of the linear epitopes recognizing mAbs. Conclusion: Our findings suggest that paired subdomains of HER2 are essential for mapping of mAbs recognizing conformational epitopes. Moreover, it seems to be no association between subdomain specificity and antitumor activity of our anti-HER2 mAbs.     

Silencing of the HER2/neu gene by siRNA inhibits proliferation and induces apoptosis in HER2/neu-overexpressing breast cancer cells

In eukaryotes, double-stranded (ds) RNA induces sequence-specific inhibition of gene expression referred to as RNA interference (RNAi). We exploited RNAi to define the role of HER2/neu in the neoplastic proliferation of human breast cancer cells. We transfected SK-BR-3, BT-474, MCF-7, and MDA-MB-468 breast cancer cells with short interfering RNA (siRNA) targeted against human HER2/neu and analyzed the specific inhibition of HER2/neu expression by Northern and Western blots. Transfection with HER2/neu-specific siRNA resulted in a sequence-specific decrease in HER2/neu mRNA and protein levels. Moreover, transfection with HER2/neu siRNA caused cell cycle arrest at G0/G1 in the breast cancer cell lines SK-BR-3 and BT-474, consistent with a powerful RNA silencing effect. siRNA treatment resulted in an antiproliferative and apoptotic response in cells overexpressing HER2/neu, but had no influence in cells with almost no expression of HER2/neu proteins like MDA-MB-468 cells. These data indicate that HER2/neu function is essential for the proliferation of HER2/neu-overexpressing breast cancer cells. Our observations suggest that siRNA targeted against human HER2/neu may be valuable tools as antiproliferative agents that display activity against neoplastic cells at very low doses.     

Spotlight on Trastuzumab in Metastatic Breast Cancer Overexpressing HER2

Trastuzumab is a humanized monoclonal antibody developed to target the HER2 receptor which is over-expressed by some cancer cells, including 25 to 30% of breast cancers. Binding with high affinity to the extracellular domain of HER2, trastuzumab inhibits the proliferation of tumor cells that overexpress HER2. A large well designed multicenter study found that the addition of trastuzumab to either an anthracycline plus cyclophosphamide or to paclitaxel, as first-line therapy for metastatic breast cancer overexpressing the HER2 receptor, significantly increased time to disease progression, rate of objective response, duration of response and survival compared with chemotherapy alone. Single-agent trastuzumab was associated with an objective response in 15% of extensively pretreated patients with metastatic breast cancer overexpressing HER2, and 26% of previously untreated patients. Patients with a HER2 overexpression level of 3+ using immunohistochemical assay, or a positive HER2 result using fluorescence in situ hybridisation, benefit more from trastuzumab therapy than those with tumors overexpressing at a level of 2+. Trastuzumab has demonstrated synergistic action with several chemotherapy agents preclinically but the optimal combination clinically is yet to be determined. Trastuzumab is generally well tolerated by most patients; the most significant adverse effects being acute fever and/or chills and the potential to cause cardiac dysfunction. Serious adverse events, including anaphylaxis and death, have occurred in 0.25% of patients. Symptomatic or asymptomatic cardiac dysfunction occurred in 27% of patients receiving an anthracycline and cyclophosphamide combined with trastuzumab. Thus, combination therapy with anthracyclines is not recommended. Symptomatic or asymptomatic cardiac dysfunction occurred in 13% of patients receiving trastuzumab plus paclitaxel and in 4.7% of patients receiving trastuzumab alone. In conclusion, intravenous trastuzumab is effective as a single-agent, and in combination with chemotherapy it significantly improves the median time to disease progression and survival time in patients with metastatic breast cancer overexpressing the HER2 receptor compared with chemotherapy alone. Cardiotoxicity is the main concern with therapy; particularly in patients with pre-existing cardiac dysfunction, the elderly and in combination with, or following, anthracyclines. Trastuzumab is indicated for use with paclitaxel as first-line therapy or as a single agent in second- or third-line treatment regimens for patients with metastatic breast cancer overexpressing HER2. Investigation is ongoing to ascertain the optimal combination regimen containing trastuzumab and antineoplastic agents. In addition, current research is focusing on the optimal timing, sequencing and duration of therapy as well as administration in the neoadjuvant and adjuvant setting.     

Inhibitory effects of combinations of HER-2/neu antibody and chemotherapeutic agents used for treatment of human breast cancers.

Previous studies have demonstrated a synergistic interaction between rhuMAb HER2 and the cytotoxic drug cisplatin in human breast and ovarian cancer cells. To define the nature of the interaction between rhuMAb HER2 and other classes of cytotoxic drugs, we applied multiple drug effect/combination index (CI) isobologram analysis to a variety of chemotherapeutic drug/rhuMAb HER2 combinations in vitro. Synergistic interactions at clinically relevant drug concentrations were observed for rhuMAb HER2 in combination with cisplatin (CI=0.48, P=0.003), thiotepa (CI=0.67, P=0.0008), and etoposide (CI=0.54, P=0.0003). Additive cytotoxic effects were observed with rhuMAb HER2 plus doxorubicin (CI=1.16, P=0.13), paclitaxel (CI=0.91, P=0.21), methotrexate (CI=1.15, P=0.28), and vinblastine (CI=1.09, P=0.26). One drug, 5-fluorouracil, was found to be antagonistic with rhuMAb HER2 in vitro (CI=2.87, P=0.0001). In vivo drug/rhuMAb HER2 studies were conducted with HER-2/neu-transfected, MCF7 human breast cancer xenografts in athymic mice. Combinations of rhuMAb HER2 plus cyclophosphamide, doxorubicin, paclitaxel, methotrexate, etoposide, and vinblastine in vivo resulted in a significant reduction in xenograft volume compared to chemotherapy alone (P<0.05). Xenografts treated with rhuMAb HER2 plus 5-fluorouracil were not significantly different from 5-fluorouracil alone controls consistent with the subadditive effects observed with this combination in vitro. The synergistic interaction of rhuMAb HER2 with alkylating agents, platinum analogs and topoisomerase II inhibitors, as well as the additive interaction with taxanes, anthracyclines and some antimetabolites in HER-2/neu-overexpressing breast cancer cells demonstrates that these are rational combinations to test in human clinical trials.     

Visualization of effective tumor targeting by CD8+ natural killer T cells redirected with bispecific antibody F(ab')(2)HER2xCD3

HER2 is an attractive immunotherapeutic target for neoplastic disease because this cell surface molecule is overexpressed on a large fraction of malignant tumor cells. To directly assess therapeutic responses to targeted therapy by noninvasive in vivo imaging in small animals, human HER2-expressing ovarian carcinoma cells were genetically modified with a firefly luciferase gene, and light emission was used for visualization of tumor growth and response to therapy. This imaging approach was able to demonstrate in real-time tumor regression in a HER2 xenograft mouse model by adoptive transfer of in vitro induced and expanded cytotoxic CD8+ natural killer T (NKT) cells retargeted with a humanized bispecific antibody F(ab')(2)HER2xCD3. Immunotherapy with effector cells alone or a humanized monoclonal antibody anti-p185(HER2) (4D5-8) resulted in significant but slower reduction in tumor burden. Long-term survival of tumor xenografts correlated inversely with visible residual tumor burden. In vitro, F(ab')(2)HER2xCD3 substantially augmented cytotoxic activity of CD8+ NKT cells. By flow-sorting, CD8+ NKT cells coexpressing CD56 were found to have the highest redirected killing ability. Treatment with concanamycin A or EGTA abrogated CD8+ NKT cytotoxicity indicating that perforin is a major pathway of tumor cell lysis. In contrast, when CD8+ NKT cell were cross-linked with F(ab')(2)HER2xCD3 neither the immunosuppressants cyclosporine A and FK506, nor the increase of intracellular cyclic AMP by dibutyryl cyclic AMP were able to inhibit cytotoxicity demonstrating that signaling via the CD3 antigen changes the biological activity of non-MHC-restricted effector cells. These studies have demonstrated that CD8+ NKT cells can be successfully redirected to tumor cells using bispecific antibodies and offer a promising strategy for adoptive immunotherapy of neoplastic diseases.     

Radionuclide therapy of HER2-positive microxenografts using a 177Lu-labeled HER2-specific Affibody molecule

A radiolabeled anti-HER2 Affibody molecule (Z(HER2:342)) targets HER2-expressing xenografts with high selectivity and gives good imaging contrast. However, the small size (approximately 7 kDa) results in rapid glomerular filtration and high renal accumulation of radiometals, thus excluding targeted therapy. Here, we report that reversible binding to albumin efficiently reduces the renal excretion and uptake, enabling radiometal-based nuclide therapy. The dimeric Affibody molecule (Z(HER2:342))(2) was fused with an albumin-binding domain (ABD) conjugated with the isothiocyanate derivative of CHX-A'-DTPA and labeled with the low-energy beta-emitter (177)Lu. The obtained conjugate [CHX-A'-DTPA-ABD-(Z(HER2:342))(2)] had a dissociation constant of 18 pmol/L to HER2 and 8.2 and 31 nmol/L for human and murine albumin, respectively. The radiolabeled conjugate displayed specific binding to HER2-expressing cells and good cellular retention in vitro. In vivo, fusion with ABD enabled a 25-fold reduction of renal uptake in comparison with the nonfused dimer molecule (Z(HER2:342))(2). Furthermore, the biodistribution showed high and specific uptake of the conjugate in HER2-expressing tumors. Treatment of SKOV-3 microxenografts (high HER2 expression) with 17 or 22 MBq (177)Lu-CHX-A'-DTPA-ABD-(Z(HER2:342))(2) completely prevented formation of tumors, in contrast to mice given PBS or 22 MBq of a radiolabeled non-HER2-binding Affibody molecule. In LS174T xenografts (low HER2 expression), this treatment resulted in a small but significant increase of the survival time. Thus, fusion with ABD improved the in vivo biodistribution, and the results highlight (177)Lu-CHX-A'-DTPA-ABD-(Z(HER2:342))(2) as a candidate for treatment of disseminated tumors with a high level of HER2 expression.     

Changes over time of extracellular domain of HER2 (ECD/HER2) serum levels have prognostic value in metastatic breast cancer

Background Blood levels of the extracellular domain of HER-2/neu (ECD/HER2) have been suggested to have potential as a tumor marker in breast cancer. Our aim was to assess the prognostic value of baseline levels of ECD/HER2, but more importantly changes in levels over time, in women with metastatic breast cancer. Methods Baseline and serial levels of ECD/HER2 were measured in 158 women with newly-diagnosed metastatic breast cancer, in whom we previously performed serial measurement of plasma osteopontin. ECD/HER2 was measured in 1,282 serum samples using a validated ELISA at baseline and every 3–12 weeks during and after therapy until death (median, n = 8 samples per patient). Multivariate time-dependent survival analyses were conducted using models that right-censored patient outcomes 3, 6 and 12 months after last known ECD/HER2 measurement. Results Thirty-four patients (22%) had elevated baseline ECD/HER2 (median 10.2 ng/ml: range 4.1–40.4 ng/ml). In univariate analysis, elevated baseline ECD/HER2 was associated with short survival (P = 0.001). In a multivariate model incorporating standard clinical prognostic factors, baseline ECD/HER2 was significantly associated with survival duration (RR 1.029; P = 0.020). Presence of visceral metastases and ECOG status 2–4 also retained significance. In a multivariate model incorporating standard prognostic factors and changes in sequential ECD/HER2 levels, an ECD/HER2 increase of >12 ng/ml at any time was the variable with most prognostic value for poor survival (RR 6.10; P = 0.0003); poor ECOG status also retained significance. Conclusion Increases over time of ECD/HER2 levels were strongly associated with poor survival in this cohort of women with metastatic breast cancer. An invited commentary to this article can be found at doi:.     

Optimizing radiolabeled engineered anti-p185HER2 antibody fragments for in vivo imaging

We have recently described the in vivo properties of an iodinated anti-p185HER2 engineered antibody fragment [minibody (scFv-C(H)3)2; 80 kDa], made from the internalizing 10H8 monoclonal antibody. Although the 10H8 minibody showed excellent binding to the target in vitro, only modest tumor uptake [5.6 +/- 1.7% injected dose per gram (ID/g) of tissue] was achieved in nude mice bearing MCF7/HER2 breast cancer tumors. Here, in an attempt to improve targeting, the 10H8 minibody was conjugated to 1,4,7,10-tetraazacyclododecane-N, N', N', N'-tetraacetic acid (DOTA), radiometal labeled, and evaluated in vivo. The tumor uptake of 111In-DOTA 10H8 minibody was 5.7 +/- 0.1% ID/g, similar to the radioiodinated 10H8 minibody. However, in addition to the expected liver clearance, the kidneys had unexpectedly high activity (34.0 +/- 4.0% ID/g). A minibody derived from a second anti-p185(HER2) antibody (trastuzumab; hu4D5v8) was also made. Tumor uptakes, evaluated by quantitative microPET using 64Cu-DOTA hu4D5v8 minibody, were 4.2 +/- 0.5% ID/g. Furthermore, in non-tumor-bearing mice, 111In-DOTA hu4D5v8 minibody exhibited similar elevated uptake in the kidneys (28.4 +/- 6.5% ID/g). Immunohistochemical staining of kidneys from non-tumor-bearing mice showed strong specific staining of the proximal tubules, and Western blot analysis of kidney lysate confirmed the presence of cross-reactive antigen. To further improve tumor uptake and normal tissue distribution, a larger hu4D5v8 fragment [(scFv-C(H)2-C(H)3)2; 105 kDa] was made, engineered to exhibit rapid clearance kinetics. This fragment, when evaluated by microPET, exhibited improved tumor targeting (12.2 +/- 2.4% ID/g) and reduced kidney uptake (13.1 +/- 1.5% ID/g). Thus, by manipulating the size and format of anti-p185(HER2) antibody fragments, the kidney activity was reduced and high or low expression of p185HER2 in xenografts could be distinguished by microPET imaging.     

Serum HER2 testing in patients with HER2-positive breast cancer: the death knell tolls

Determination of the human epidermal growth factor receptor 2 (HER2; also known as ERBB2) status of breast tumours is emphasised in various national guidelines as a necessary step for the diagnosis of breast cancer. As an alternative to tissue-based diagnostic methods, there has been substantial interest in the establishment of an easily accessible serum-based alternative that could be used for prognosis and diagnosis. Detection of serum-soluble-HER2 extracellular domain (ECD) and establishment of its potential clinical usefulness has created much debate. We assessed whether identification of circulating concentrations of HER2 ECD have clinical usefulness for management of patients with HER2-positive breast cancer. We examined data from 63 studies of patients with breast cancer. Prevalence of increased concentrations varied greatly between studies. Some studies showed significant associations between raised concentrations and poor prognosis, poor response to treatments including trastuzumab, or tumour characteristics associated with aggressive disease, whereas others did not. Examination of existing data showed that concentrations of HER2 ECD are not consistently related to patient outcomes; therefore, there is insufficient evidence to support the clinical use of serum HER2 ECD testing. Design and execution of future large-scale trials to investigate the clinical use of HER2 ECD testing, in view of the progressive non-supportive evidence, is not recommended. Oncologists should continue to adhere to national guidelines for determining HER2 status. Furthermore, oncologists should continue to use clinical parameters when making decisions about initiation, continuation, and discontinuation of HER2-targeted treatments.